CN107202884A - Based on immunomagnetic beads and MnO2Nano-particle detects vibrio parahemolyticus kit - Google Patents

Based on immunomagnetic beads and MnO2Nano-particle detects vibrio parahemolyticus kit Download PDF

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CN107202884A
CN107202884A CN201710459663.3A CN201710459663A CN107202884A CN 107202884 A CN107202884 A CN 107202884A CN 201710459663 A CN201710459663 A CN 201710459663A CN 107202884 A CN107202884 A CN 107202884A
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vibrio parahemolyticus
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CN107202884B (en
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赵超
刘玉申
李娟�
王娟
付凯悦
聂玮
宋秀玲
徐坤
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Jilin University
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    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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Abstract

The invention discloses based on immunomagnetic beads and MnO2Nano-particle detects vibrio parahemolyticus kit, prepares the magnetic bead with superparamagnetism, and the magnetic bead and vibrio parahemolyticus polyclonal antibody are coupled；Synthesize MnO2Nano-particle, the nano-particle and vibrio parahemolyticus specific egg yolk antibody are coupled；Prepare liquid is taken, it is mixed with two kinds of probes, magnetic bead thalline MnO is separated using magnetic frame2Compound, the mixture is resuspended with citrate buffer, TMB colour developings is added, so as to realize the quick specific detection of vibrio parahemolyticus.The invention proposes to utilize immunomagnetic beads and MnO2Nano-particle technology is detected to vibrio parahemolyticus, is developed the color using the immunological response of ELISA method, is shortened detection time, the coefficient of variation is small when quantitatively detecting.Minimal detectable concentration is 10CFU/mL, and recovery of standard addition reaches 96.7%, and sensitivity is high, and stability is good.

Description

Based on immunomagnetic beads and MnO2Nano-particle detects vibrio parahemolyticus kit
Technical field
The invention belongs to microorganism detection field, and in particular to based on immunomagnetic beads and MnO2Nano-particle detects parahemolyticas Vibrios kit.
Background technology
Vibrio parahemolyticus（Vibrio parahaemolyticus, VP）It is a kind of non-gemma Halophiles of Gram-negative, It is one of common food-borne pathogens.The bacterium is mainly derived from shrimp, the marine product such as fish and shellfish, mainly passes through the sea of pollution Product causes acute human gastroenteritis, wound infection and septicemia etc..In recent years, as the crowd of edible marine product is continuous Amplification, food poisoning is in rising trend as caused by vibrio parahemolyticus, it was reported that since nineteen ninety-eight, vibrio parahemolyticus Caused food poisoning exceedes Salmonella food poisoning, the foodborne bacterial pathogenses as most serious.At present, the bacterium is that majority enters The pathogenic bacteria that outlet aquatic products must be examined.
Currently, the traditional detection method of China vibrio parahemolyticus is based on culture of microorganism, and whole process probably needs 5-8 My god, cumbersome, time-consuming, and the fast and convenient requirement of the food-safe detection of people can not have been met.It is continuous with technology It is progressive, promote continuing to develop for detection method, increasing method is applied to detection vibrio parahemolyticus, such as round pcr, Enzyme linked immunosorbent assay（ELISA）, loop-mediated isothermal amplification technique（LAMP）Deng.Round pcr can amplification detection in a short time Target, with sensitivity it is high the characteristics of, but PCR operating process requires high and false positive easily occurs.ELISA method using antigen- The specific binding detection of antibody, it is low for equipment requirements, but time-consuming, sensitivity is comparatively relatively low.LAMP is used as one Emerging detection technique, low with expense, sensitivity is high, the features such as specific high, but LAMP needs first selective enrichment training Support, therefore be unfavorable for the Site Detection of sample, and be likely to enter new pollution.So, it is very necessary to have exploitation a kind of fast Speed, sensitivity is high, the detection means of high specificity, to monitor food-borne pathogens, to ensure the food security of people.
The content of the invention
It is an object of the present invention to provide a kind of quick, sensitive, easy vibrio parahemolyticus detection based on immunomagnetic beads and MnO2Nano-particle detects vibrio parahemolyticus kit.
Based on immunomagnetic beads and MnO2Nano-particle detects vibrio parahemolyticus kit, and it includes：Carboxylated Fe3O4Receive Rice corpuscles and the immunomagnetic beads and MnO of the anti-how anti-coupling of vibrio parahemolyticus rabbit2Nano-particle and anti-vibrio parahemolyticus are special Property chicken yolk antibody IgY coupling MnO2Nano-probe.
The method of quick detection food vibrio parahemolyticus, it includes：
1）The preparation of nanometer magnetic bead：
Add 1.08g FeCl3.6H2In O to 22mL ethylene glycol, ultrasonic dissolution 10min,；1.2g NaAc and 0.2g lemons are added again Lemon acid sodium ultrasound 10min,；Finally, addition 0.2g PEG6000 are in above-mentioned solution, and ultrasound is mixed, and gained homogeneous solution is turned Move in reactor, 18h is reacted in 199 DEG C of baking ovens；The solid matter of black in being separated with permanent magnet from reaction solution, is used Alternately cleaning 3-5 times of ultra-pure water and ethanol, obtains carboxylated Fe3O4Nano-particle；
2）The preparation of immunomagnetic beads：
Take 0.5mg carboxylated Fe3O4Nano-particle, is washed 2 times with PBS, is resuspended with 1mL PBS, adds 10mg EDC and 10mg After NHS activated carboxyls, 30min, magnetic suck removes supernatant, and PBS is washed 3 times, and 1mL PBS are resuspended, and adds the 50 anti-vibrio parahemolyticus of μ g Rabbit resists more, reacts at room temperature 2h, and Magneto separate removes supernatant, and PBS is washed 3 times, adds 1mL confining liquids closing 1h, obtains functionalization and magnetic is immunized Pearl；
3）Nanometer MnO2The preparation of particle：
25mg bovine serum albumin(BSA)s are added in 10mL PBS, are stirred and evenly mixed, 100 μ L MnSO are added4.H2O (0.1M) is in mixing In liquid, stir 2min, then to its add 1M the μ L of NaOH 100, stirring 6-7h after, with ultra-pure water dialyse 48h, 15000rpm from The heart, obtains MnO2Nano-particle；
4）MnO2The preparation of nano-probe：
Take 1mg MnO2Nano-particle, PBS is resuspended, and adds 1mg EDC and 1.5mg NHS activation 15min, adds the anti-pairs of 50 μ g molten Courageous and upright vibrios specific egg yolk antibody IgY, reacts 2h, and 15000 rpm centrifugations obtain functionalization MnO2Nano-probe；
5）The detection of vibrio parahemolyticus：
Take analyte sample fluid 100 μ L, MnO2Nano-probe 100 μ L, immunomagnetic beads 0.5mg resuspension is 800 μ L in room temperature reaction 30min, Magneto separate removes supernatant, and PBS is washed 3 times, 1mL citrate buffers and 10 μ L TMB colour developing 10min is added, in ultraviolet Spectrophotometric measures absorption spectrum；
Described PBS is 8g NaCl, 0.2g KCl, 3.63g Na2HPO4.12H2O, 0.24g KH2PO41L is dissolved in surpass In pure water, pH to 7.4 is adjusted；
Described confining liquid is 10mg bovine serum albumin(BSA)s（BSA）Add in 1mL PBS buffer solutions, matching while using；
Described citrate buffer is 0.73g Na2HPO4.2H2O, 0.4665g citric acid are dissolved in 50mL ultra-pure waters, are adjusted Save pH to 5；
Described step 1）Fe3O4Nano-particles size is 130-330nm.
The invention provides based on immunomagnetic beads and MnO2Nano-particle detects vibrio parahemolyticus kit, and preparation has The magnetic bead of superparamagnetism, the magnetic bead and vibrio parahemolyticus polyclonal antibody are coupled；Synthesize MnO2Nano-particle, by the nanometer Particle is coupled with vibrio parahemolyticus specific egg yolk antibody；Prepare liquid is taken, it is mixed with two kinds of probes, magnetic frame is used Separate magnetic bead-thalline-MnO2Compound, the mixture is resuspended with citrate buffer, TMB colour developings is added, so as to realize pair The quick specific detection of hemolytic vibrios.The invention proposes to utilize immunomagnetic beads and MnO2Nano-particle technology is to parahemolyticas arc Bacterium detects, is developed the color using the immunological response of ELISA method, shortens detection time, the coefficient of variation is small when quantitatively detecting.It is minimum Detectable concentration is 10CFU/mL, and recovery of standard addition reaches 96.7%, and sensitivity is high, and stability is good.
Brief description of the drawings
Fig. 1 immunomagnetic beadses and MnO2Overhaul flow chart of the nano-probe to vibrio parahemolyticus.
Embodiment
The vibrio parahemolyticus rabbit clonal antibody IgG of embodiment 1 preparation
It is 1 × 10 to take concentration9 CFU·mL-1Freund's complete adjuvant/incomplete Freund's adjuvant of inactivated bacterial liquid and equivalent has been emulsified Entirely, inactivated vaccine is prepared.First immunisation takes concentration to be 1 × 109 CFU·mL-1Freund's complete adjuvant vaccine immunity health New Zealand White Rabbit（There is provided by preclinical medicine institute of Jilin University Experimental Animal Center）, using dorsal sc multi-point injection method, every immune 2 ML, in second week, the 3rd week incomplete Freund's adjuvant vaccine using Isodose is immune for the second time to rabbit progress, exempt from for the third time Epidemic disease.After 10 days, with inactivated bacterial liquid booster immunization twice.Immune preceding rabbit auricular vein takes blood every time, determines serum titer.Reach point From standard, rabbit heart blood sampling separates serum, obtains the antiserum of anti-vibrio parahemolyticus.Using saturated ammonium sulfate salt precipitation Method is purified to the polyclonal antibody IgG in described antiserum；Using indirect elisa method to the potency of purified antibodies and Specificity is measured, and table 1 is immune front and rear rabbit anteserum and antibody titer measurement result, is as a result shown, with immune time Increase, serum antibody titer gradually rises, and the vibrio parahemolyticus rabbit polyclonal antibody potency finally obtained can reach 1: 1024000；Table 2 is rabbit polyclonal antibody IgG specific outcomes, is as a result shown, the antibody specificity of preparation is preferable；Using BCA Protein quantification kit is measured to the concentration of IgG antibody after purification, and is adjusted to its protein concentration with PBS 1mg/mL, -20 DEG C save backup.
The vibrio parahemolyticus chicken yolk antibody IgY of embodiment 2 preparation
It is 1 × 10 to take concentration9 CFU·mL-1Freund's complete adjuvant/incomplete Freund's adjuvant of inactivated bacterial liquid and equivalent has been emulsified Entirely, inactivated vaccine is prepared, for Immune Laying Hens.By vaccine inoculation in pigeon breast by the way of muscle multi-point injection.First immunisation Using Freund's complete adjuvant vaccine, every chicken 1mL, interval is exempted from for the second time after two weeks using incomplete Freund's adjuvant vaccine Epidemic disease, then carried out primary reinforcement every two weeks and is immunized.Collect and front and rear egg is immunized, be stored in standby in 4 DEG C of refrigerators.Using The saltings out method of PEG 6000 are extracted to the Yolk antibody in egg.Using indirect elisa method to rear Yolk antibody before purification Potency and specificity are measured, and table 3 is chicken serum and antibody IgY titration result before and after immune, is as a result shown, with exempting from Antibody titer in the increase of epidemic disease number of times, chicken serum gradually rises, and after last time is immune, antibody titer is up to 1：64000, warp After extraction purification, the vibrio parahemolyticus IgY antibody titers finally obtained can reach 1:128000.Table 4 is chicken yolk antibody IgY specific outcomes, as a result show that the IgY antibody specificities of preparation are preferable；Using BCA protein quantifications kits to after purification The concentration of IgY antibody is measured, and its protein concentration is adjusted into 1mg/mL with PBS, and -20 DEG C save backup.
The preparation of the immunomagnetic beads of embodiment 3
Add 1.08g FeCl3.6H2In O to 22mL ethylene glycol, ultrasonic dissolution 10min forms red tan solution；Add again 1.2g NaAc and 0.2g sodium citrate are into the solution, ultrasonic 10min, form dark red solution；Finally, 0.2g is added PEG6000 is in above-mentioned solution, and ultrasound is mixed, and gained homogeneous solution is transferred in reactor, is reacted in 199 DEG C of baking ovens 18h.The solid matter of black in being separated with permanent magnet from reaction solution, with ultra-pure water and ethanol alternately cleaning 3-5 times, is obtained Fe3O4Nano-particle；Take 0.5mg carboxylated Fe3O4Nano-particle, is washed 2 times with PBS, is resuspended with 1mL PBS, adds 10mg EDC With 10mg NHS activated carboxyls, after 30min, magnetic suck removes supernatant, and PBS is washed 3 times, and 1mL PBS are resuspended, and adds the anti-pairs of 50 μ g molten Courageous and upright vibrios rabbit polyclonal antibody, reacts at room temperature 2h, and Magneto separate removes supernatant, and PBS is washed 3 times, adds 1mL confining liquids closing 1h, obtains To functionalization immunomagnetic beads.
The MnO of embodiment 42The preparation of nano-probe
Add 25mg bovine serum albumin(BSA)s（BSA）In 10mL PBS, stir and evenly mix, add 100 μ L MnSO4.H2O(0.1M) In mixed liquor, 2min is stirred, solution becomes milky, then adds 100 μ L NaOH (1M) to it, and solution is by milky gradual change For brown color, stir after 6-7h, with ultra-pure water dialysis 48h, 15000rpm centrifugation, obtain MnO2 nano-particles；Take 1mg MnO2 Nano-particle, PBS is resuspended, and adds 1mg EDC and 1.5mg NHS activation 15min, adds the anti-vibrio parahemolyticus specificity of 50 μ g Chicken yolk antibody（IgY）, 2h is reacted, 15000rpm centrifugations obtain functionalization MnO2 nano-probes.
The configuration of the buffer solution of embodiment 5
PBS:Take 8g NaCl, 0.2g KCl, 3.63g Na2HPO4.12H2O, 0.24g KH2PO4It is dissolved in 1L ultra-pure waters In, adjust pH to 7.4.
The preparation of the bacterium solution standard items of embodiment 6
- 80 DEG C of preservation strains are taken, bacterial strain activation is carried out.Bacterial strain after activation is drawn on 3% sodium chloride tryptone agar flat board Line point is pure, and after 37 DEG C of 18 ~ 24 h of culture, picking single bacterium colony is inoculated with 3% sodium chloride tryptone fluid nutrient medium, 37 DEG C are shaken Swing the h of culture 12 ~ 18.10 times of doubling dilution flat board tilt-pour processes of 1mL bacterium solutions are taken to carry out count plate.Remaining bacterium solution is with 1 % formaldehyde 10 min are inactivated at room temperature, the rpm of bacterium solution 3000 of inactivation is centrifuged into 3 min, are collected thalline, are then filled with PBS solution Divide and mix, bacteria suspension is made simultaneously, it is 10 to adjust bacteria suspension concentration with PBS solution9 CFU·mL-1, it is stored in standby in 4 DEG C of refrigerators.
Embodiment 7 is based on immunomagnetic beads and MnO2The method that nano-particle detects vibrio parahemolyticus
Overhaul flow chart such as Fig. 1.Take analyte sample fluid 100 μ L, MnO2800 μ are resuspended in nano-probe 100 μ L, immunomagnetic beads 0.5mg L removes supernatant in room temperature reaction 30min, Magneto separate, and PBS is washed 3 times, adds 1mL citrate buffers and 10 μ L TMB colour developings 10min, absorption spectrum is surveyed in ultraviolet specrophotometer.With reference to the canonical plotting done, vibrio parahemolyticus in sample is determined Quantity.Quantitatively detect the scope of the bacteria concentration in 10-105CFU/mL. the inventive method detection is stable, and test limit can as little as 10 CFU/mL, detection time is short, and Detection results are good.To vibrio parahemolyticus, listeria monocytogenes（LM）, will congratulate Bacterium, staphylococcus aureus, salmonella, Escherichia coli O 157：The bacterium such as H7 are detected, the only detection of vibrio parahemolyticus As a result it is positive, remaining is feminine gender, this method high specificity has no false positive and false negative result, as a result such as table 5.
Note：<+>Represent that vibrio parahemolyticus is positive,<->Represent that vibrio parahemolyticus is negative.
The preparation of the kit of embodiment 8
As the anti-vibrio parahemolyticus immunomagnetic beads described by embodiment 3, the MnO described by implementation 42Nano-probe, implementation The bacterium solution standard items described by buffer solution, embodiment 6 described by example 5 are collectively constituted based on immunomagnetic beads and MnO2Nano-particle Detect the kit of vibrio parahemolyticus.
The detection of the analog sample of embodiment 9
Prepare clam leachate：It is accurate to weigh 5g clam meat, 5mL sterilizing PBS are added, after grinding is uniform, is filtered, used with filter paper 0.22 μm of filter membrane carries out suction filtration to filter liquor；The vibrio parahemolyticus of various concentrations is taken to be inoculated in the leachate, with the present invention Detection method is detected to it.Take analyte sample fluid 100 μ L, MnO2Nano-probe 100 μ L, immunomagnetic beads 0.5mg are resuspended 800 μ L remove supernatant in room temperature reaction 30min, Magneto separate, and PBS is washed 3 times, add 1mL citrate buffers and 10 μ L TMB colour developings 10min, absorption spectrum is surveyed in ultraviolet specrophotometer.
With reference to the canonical plotting done, the quantity of vibrio parahemolyticus in sample is determined.Quantitatively detect the bacteria concentration Scope is in 10-103CFU/mL. the inventive method detection is stable, and recovery of standard addition reaches 96.7%.

Claims (6)

1. based on immunomagnetic beads and MnO2Nano-particle detects vibrio parahemolyticus kit, and it includes：Carboxylated Fe3O4Nanometer Particle and the immunomagnetic beads and MnO of the anti-how anti-coupling of vibrio parahemolyticus rabbit2Nano-particle and anti-vibrio parahemolyticus specificity The MnO of chicken yolk antibody IgY couplings2Nano-probe.
2. the method for quick detection food vibrio parahemolyticus, it includes：
1）The preparation of nanometer magnetic bead：
Add 1.08g FeCl3.6H2In O to 22mL ethylene glycol, ultrasonic dissolution 10min,；1.2g NaAc and 0.2g lemons are added again Lemon acid sodium ultrasound 10min,；Finally, addition 0.2g PEG6000 are in above-mentioned solution, and ultrasound is mixed, and gained homogeneous solution is turned Move in reactor, 18h is reacted in 199 DEG C of baking ovens；The solid matter of black in being separated with permanent magnet from reaction solution, is used Alternately cleaning 3-5 times of ultra-pure water and ethanol, obtains carboxylated Fe3O4Nano-particle；
2）The preparation of immunomagnetic beads：
Take 0.5mg carboxylated Fe3O4Nano-particle, is washed 2 times with PBS, is resuspended with 1mL PBS, adds 10mg EDC and 10mg NHS After activated carboxyl, 30min, magnetic suck removes supernatant, and PBS is washed 3 times, and 1mL PBS are resuspended, and adds the anti-vibrio parahemolyticus rabbits of 50 μ g Resist more, react at room temperature 2h, Magneto separate removes supernatant, and PBS is washed 3 times, add 1mL confining liquids closing 1h, obtain functionalization and magnetic is immunized Pearl；
3）Nanometer MnO2The preparation of particle：
25mg bovine serum albumin(BSA)s are added in 10mL PBS, are stirred and evenly mixed, 100 μ L MnSO are added4.H2O (0.1M) is in mixing In liquid, stir 2min, then to its add 1M the μ L of NaOH 100, stirring 6-7h after, with ultra-pure water dialyse 48h, 15000rpm from The heart, obtains MnO2Nano-particle；
4）MnO2The preparation of nano-probe：
Take 1mg MnO2Nano-particle, PBS is resuspended, and adds 1mg EDC and 1.5mg NHS activation 15min, adds the anti-pairs of 50 μ g molten Courageous and upright vibrios specific egg yolk antibody IgY, reacts 2h, and 15000 rpm centrifugations obtain functionalization MnO2Nano-probe；
5）The detection of vibrio parahemolyticus：
Take analyte sample fluid 100 μ L, MnO2Nano-probe 100 μ L, immunomagnetic beads 0.5mg resuspension is 800 μ L in room temperature reaction 30min, Magneto separate removes supernatant, and PBS is washed 3 times, 1mL citrate buffers and 10 μ L TMB colour developing 10min is added, in ultraviolet Spectrophotometric measures absorption spectrum.
3. the method for quick detection food vibrio parahemolyticus according to claim 2, it is characterised in that：Described PBS Buffer solution is 8g NaCl, 0.2g KCl, 3.63g Na2HPO4.12H2O, 0.24g KH2PO4It is dissolved in 1L ultra-pure waters, adjusts pH To 7.4.
4. the method for the quick detection food vibrio parahemolyticus according to Claims 2 or 3, it is characterised in that：Described Confining liquid is 10mg bovine serum albumin(BSA)s（BSA）Add in 1mL PBS buffer solutions, matching while using.
5. the method for quick detection food vibrio parahemolyticus according to claim 4, it is characterised in that：Described lemon Lemon phthalate buffer is 0.73g Na2HPO4.2H2O, 0.4665g citric acid are dissolved in 50mL ultra-pure waters, adjust pH to 5.
6. the method for quick detection food vibrio parahemolyticus according to claim 5, it is characterised in that：Described step 1）Fe3O4Nano-particles size is 130-330nm.
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CN110470840A (en) * 2019-09-06 2019-11-19 齐鲁工业大学 A method of detection vibrio parahemolyticus
CN110850076A (en) * 2019-11-28 2020-02-28 南京迪安医学检验所有限公司 Anti-double-chain DNA antibody IgG chemiluminescence immunoassay kit and preparation method and application thereof
CN111323596A (en) * 2020-03-11 2020-06-23 赵薇 Staphylococcus aureus detection kit and preparation method thereof
CN111575299A (en) * 2020-05-11 2020-08-25 吉林农业大学 Preparation method of bovine capsular A type pasteurella multocida immunomagnetic beads
CN112557384A (en) * 2020-12-10 2021-03-26 贵州大学 Hydrogen sulfide detection method based on colorimetric analysis and application
CN112649602A (en) * 2020-06-17 2021-04-13 吉林大学 Visual kit for detecting staphylococcus aureus based on immunomagnetic beads
WO2023025074A1 (en) * 2021-08-25 2023-03-02 中国农业科学院油料作物研究所 Immunoassay kit having multiple test scales, and application thereof
CN117110607A (en) * 2023-09-16 2023-11-24 鲁东大学 Composition for detecting vibrio parahaemolyticus and application thereof

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